A barrier to increasing human understanding of cellular biology may be that current techniques do not permit the visualization of proteins and cell structures in a way that allows a viewer to understand the operation of proteins in a cell. For example, technology such as fluorescence light microscopy may be widely used for protein localization with a cell, but subcellular context, such as organelles are often absent in images produced by fluorescence light microscopy. On the other hand, electron microscopy can map membranes of a cell, which in turn makes organelles inside the cell visible, but electron microscopy is limited in the ability to specifically localize proteins.
Correlative approaches may allow imaging a specimen with fluorescence light microscopy or electron microscopy, but may lack an effective process to correlate the fluorescence light microscopy image with the electron microscopy image. The low resolution of fluorescence images often precludes the precise localization of proteins in relation to specific organelles or microenvironments within a cell. Advances in sub diffraction resolution fluorescence light microscopy techniques, such as fluorescence photoactivation localization microscopy (FPALM), photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction (dSTORM) may resolve protein distributions more than ten times better than conventional light microscopy. These advances in sub diffraction resolution fluorescence light microscopy have triggered the development of new correlative approaches such as nano resolution fluorescence electron microscopy (Nano-fEM). Nano-fEM localizes proteins at the nanoscale by imaging a cell sample using sub diffraction resolution localization microscopy (FPALM/dSTORM) and electron microscopy, and the datasets produced by Nano-fEM may be manually correlated to provide a visualization of protein distribution within a cell.